The long term objectives are to gain a greater understanding of post- replicative DNA modifications and the enzymes that mediate them with respect to their nature, mechanism, specificity, biological function and regulation. To this end, two systems have been studied in detail: (i) regulation of the phage Mu mom operon; and (ii) the Dam DNA-[N6-adenine] methyltransferases of phages T2 and T4. In the phage Mu system, expression of mom-modification is regulated at the level of transcription and translation; in contrast, nothing is known about the modification process itself. The genes for transactivation factors, C and Com, have been cloned and sequenced, and the proteins purified. One important set of aims is to elucidate the interactions with their target molecules in order to understand the mechanism by which they activate transcription or translation, respectively. Because Com is known to specifically bind a site on com-mom mRNA, it is formally analogous to the Human Immunodeficiency Virus Type 1 (= HIV-1) Tat and Rev proteins, each of which binds (along with host factors) to a specific site on HIV mRNA. since the Mu system is less complex, insights into Com function might be applicable to understanding the HIV-1 proteins. The small size of com and C, and the ability to do genetic, biochemical and physical-chemical analyses, make them exquisite systems for studying protein-nucleic acid interactions. Collaborative efforts will be undertaken to determine the 3-D structures of Com and C. We will also investigate the DNA-'momification' process itself and why it is lethal to the host cell. Altered forms of the T4 dam gene will be produced by site-directed mutagenesis and the mutant enzymic forms purified according to procedures which we have developed for the wild-type protein. They will be studied in order to delineate the functional domains for recognition/binding of substrate DNA and S-adenosyl-methionine and the nature of DNA methyltransferase-substrate interactions. Through collaborative efforts the 3-D structure of the Dam protein will be determined. The T2 and T4 forms of Dam, differing in only three amino acids also differ in their ability to methylate non-canonical sites (sequences other than GATC). Through a combination of recombinant DNA technology (to produce chimeric and mutant forms of Dam) and enzyme kinetics analysis, we will determine which of the three residues is responsible for this property. Because the T2 dam gene has 536 nucleotides located just 5' to its promoter, dam expression may be different following infection with T2 and T4. Therefore, a variety of techniques will be applied to measure the post- infection levels of Dam protein and mRNA.